There is a gap in our understanding of how the white, brown, and beige adipocyte subtypes are programmed to adopt their distinct phenotypic characteristics of storing or burning energy. The imbalance between energy intake and expenditure can lead to obesity and metabolic disorders such as type 2 diabetes. White adipocytes store and mobilize energy for peripheral tissue consumption, while brown adipocytes store and burn energy during cold exposure to generate heat. Beige adipocytes behave like brown adipocytes, but appear in white adipose tissue with long-term cold exposure. Our long-term objective is to better understand how adipocyte precursors are programed to adopt these distinct phenotypic characteristics. Ultimately our goal is to shift the energy balance from storage to expenditure. To this end we have made significant progress in completing our studies described in the KO1 award where we have determined that TLE3 and Prdm16 drive opposing transcriptional programs to stimulate white or brown fat gene expression, respectively. In this RO3 application, we will extend our findings from the KO1 award to gain a molecular understanding of how TLE3 and Prdm16 counter each other's actions. Our hypothesis is that TLE3 directly interacts with Prdm16 to form a mutually neutralized coactivator complex to regulate energy storage or expenditure. Where a bound TLE3-Prdm16 complex is inactive, and unbound TLE3 or Prdm16 are free to interact with PPAR to stimulate energy storage or energy expenditure, respectively. We expect that this interaction will be most important in beige adipocytes where TLE3 and Prdm16 expression is high. Guided by strong preliminary data, this hypothesis will be tested in Aim1 where we will determine the functional significance of the TLE3-Prdm16 interaction, and in Aim2 where we will determine whether TLE3 blocks the appearance of beige adipocytes. Under Aim1, we've engineered several TLE3 and Prdm16 deletions to map the domains that mediate the TLE3-Prdm16 interaction and have developed a yeast-two-hybrid system to identify point mutations that will allow us to test the biological significance of the TLE3-Prdm16 interaction. Under Aim2, we will utilize our unique gain and loss of function mouse models to test whether TLE3 blocks the cold-induced appearance of beige cells in subcutaneous adipose tissue. Understanding the transcriptional mechanisms that distinguish between the adipocyte subtypes will be key to identifying novel therapeutic targets to program cells to adopt the favorable brown/beige adipocyte phenotype to treat obesity.